In this example, the sequence of events for producing a targeted mutant are shown. The binding specificity of the designed zincfinger domain directs the zfn to a specific genomic site. The plant genome original research genome engineering of. Dna fingerprinting by restriction endonuclease digestion. Zincfinger nucleases zfns drive efficient genome editing by introducing a doublestrand break into the targeted gene.
A novel zincfinger nuclease platform with a sequence. Although more than 30 singlestrandspecific nucleases from various sources. Probing isoformspecific functions of polypeptide galnac. Zfninduced doublestrand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies. Simultaneous zincfinger nuclease editing of the hiv. The oglycoproteome is differentially regulated in cells by dynamic expression of a subset of 20 polypeptide galnactransferases galnacts, and methods to identify important functions of individual galnacts are largely unavailable. Choose from 37 different sets of nuclease flashcards on quizlet. Christensen3, benjamin dake4, abirami lakshmanan1, charlotte kuperwasser4, gary d. Zinc finger nuclease zfn license agreement sigmaaldrich.
Depending on the type of zfn technology deployed, mutations can either be restricted to one or a few nucleotides or involve the insertion of a. Microinjection and homologous recombination inembryonic stem es cells, are robust but overallinefficient. Members of the zinc finger consortium continue to work to develop robust, publicly available methods for engineering zinc finger nucleases that function well in various cellular environments. Diversifying the structure of zinc finger nucleases for highprecision. Zincfinger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools.
Single strand specific nucleases pdf stranded nucleic acids and singlestranded regions in doublestranded nucleic. New plant breeding techniques food standards australia. The generation of genetically modified animals or plants with genetargeted deletions or modifications is a powerful tool to analyze gene function, study disease and produce organisms of economical interest. Even purified water can have a high ph and minerals. These enzymes can be used in a wide variety of applications, including nextgeneration sequencing ngs, pcr and gene synthesis. Scientific opinion addressing the safety assessment of. Faster method of engineering zincfinger nucleases developed. The mrna or dna encoding a zincfinger nuclease zfn, a transcription activatorlike effector nuclease talen, or a clustered regularly interspaced short palindromic repeatcrisprassociated crisprcas system, and a repair template could potentially be introduced into. Zinc finger protein motif that forms a compact globular structure that coordinates one or more zn ions. Targetable nucleases, including zincfinger nucleases zfns, transcription activatorlike effector nucleases talens, and clustered regularly interspaced short palindromic repeat. Researchers have developed a faster way to engineer synthetic enzymes that target specific dna sequences for inactivation, repair or alteration. The compozr custom zincfinger nuclease zfn service enables precise genome editing in any organism or cell line at any locus defined by the user. Other procedures can utilize either 1finger or 2finger modules to generate zincfinger arrays with six or more individual zinc fingers.
Zinc finger nucleases are useful to manipulate the genomes of many plants and animals. Targeted genome modification in mice using zincfinger nucleases. Wilen, 1 jianbin wang, 2 jennifer duong, 2 anthony j. The dnarna duplex formed between a chimeric 2frnadna. As the archetypal platform for programmable dna cleavage, zincfinger nucleases zfns have had a central role in the development and. Genome engineering with zinc finger nuclease and talens with the advances in largescale genome sequencing, it is now possible to sequence whole genomes quite readily. Scientific opinion addressing the safety assessment of plants developed using zinc finger nuclease 3 and other sitedirected nucleases with similar function1 efsa panel on genetically modified organisms gmo2, 3 european food safety authority efsa, parma, italy abstract. In living organisms, they are essential machinery for many aspects of dna repair. A new case has just come in this morning requiring your considerable expertise. Dnabinding zinc finger proteins zfps and a nuclease. Using defined fingerfinger interfaces as units of assembly for constructing zincfinger nucleases cong zhu1, ankit gupta1,2, victoria l.
Nuclease contamination in reagents used for nucleic acid isolation and analysis can contribute to experimental inconsistency, and sometimes even experimental failure. A zfn designed to create a dna doublestrand break dsb in the target locus is composed of two. Ascending dose study of genome editing by the zinc finger. The potential of gene targeting with zinc finger nucleases zfns was first explored by the groups of carroll and. In trying to pinpoint the sources of contamination, it is easy to overlook the water used in the experiment, either to prepare reagents, or to resuspend precipitated rna. Genome editing with modularly assembled zincfinger nucleases. Genome engineering with zinc finger nuclease and talens.
Genome editing with engineered zinc finger nucleases. Genome editing with compozr custom zinc finger nucleases. Sb9 is an intravenously delivered zinc finger nuclease zfn therapeutic for genome editing. The purpose of the study is to evaluate the safety, tolerability and effect on leukocyte and plasma iduronate 2sulfatase ids enzyme activity of ascending doses of sb9. The dna binding motif specified by the zinc fingers directs the zfn to a specific targeted locus in the genome.
It can also modify certain existing characteristics. A novel zincfinger nuclease platform with a sequencespecific cleavage module benno schierling1, nadine dannemann2, lilia gabsalilow1, wolfgang wende1, toni cathomen2 and alfred pingoud1 1institute of biochemistry, justusliebig university, heinrichbuffring 58, d35392 giessen and 2institute of experimental hematology, hannover medical school, carlneubergstrasse 1, d30625 hannover. Zinc finger engineering the zinc finger consortium. A nuclease also archaically known as nucleodepolymerase or polynucleotidase is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules.
Mammalian gene targeting with designed zinc finger nucleases. All members have committed to making all protocols, software, dna sequences, and reagents they publish available to the academic scientific community. Zincfinger nuclease dimers are more difficult to engineer than single dnabinding domains. Zinc finger nucleases were also generated that stimulated gene targeting at full sites from the. These artificial nucleases are powerful tools for genome modification, as they can generate a sitespecific doublestrand break dsb within the genome to promote a number of different types of genome editing 2, 3. Alternatively, repair of a dsb by homologydirected repair with an exogenously introduced donor template can promote efficient introduction of.
Zfns are also used to create a new generation of genetic disease models called isogenic human disease models. The european commission requested that the efsa panel on genetically modified organisms deliver a scientific opinion related to risk assessment of plants developed using the zinc finger nuclease 3 technique zfn3 which allows the integration of genes in a predefined insertion site in the genome of the recipient species. Zinc finger nucleases as tools to understand and treat human. Zincfinger nucleases zfns are chimeric fusions between a programmable zincfinger array zfa and the nuclease domain of foki 1. You may not transfer the product, its components, or any materials made through the use of this product, including modified animals, to any third party without prior written approval of sigma and without the transferee entering into a material transfer agreement with sigma. Dna fingerprinting by restriction endonuclease digestion an assault in east lansing you are currently employed by the michigan state police as a forensic dna analyst at their lansing laboratory. The zinc fingers are part of the bindingdomain and have precisely identified and bound to a group of matching nucleobases in the dna sequence.
However, recent advances in generating customized zincfinger nucleases zfns that can create a dna doublestrand break dsb at preselected sites in the. Repair of nucleaseinduced doublestranded breaks dsbs by nonhomologous endjoining nhej or homologydirected repair hdr can. Dimeric crispr rnaguided foki nucleases for highly. Nuclease protection assay is a laboratory technique used in biochemistry and genetics to identify individual rna molecules in a heterogeneous rna sample extracted from cells. For the generation of a novel and highly specific chimeric nuclease, a threefinger zf dnabinding protein as specific dnabinding module and the sequencespecific restriction endonuclease pvuii as dnacleavage module were fused via a short linker. Zinc finger nucleases zfns are artificial restriction enzymes made by fusing reprogrammable zincfinger dnabinding units to the foki nuclease domain, which efficiently induce, sitespecific. Sitedirected nuclease technology can be used to precisely remove undesirable traits in plants such as antinutrients or allergens, in order to reduce environmental pollution or to enhance the nutritional value of a crop, for instance in maize. Induction of doublestrand breaks by s1 nuclease, mung. Zincfinger nuclease zfn technology involves the use of an engineered zinc finger nuclease to introduce sitespecific mutations into the plant genome. Zfn dimers induce targeted dna dsbs that stimulate dna damage response pathways. The most common modular assembly process involves combining three separate zinc fingers that can each recognize a 3 basepair dna sequence to generate a 3finger array that can recognize a 9 basepair target site. Engineered zincfinger nucleases zfns can be used to introduce targeted alterations into genomes of model organisms, plants, and human cells. Restricted spacer tolerance of a zinc finger nuclease with a six amino acid linker. Zinc finger nucleases for targeted mutagenesis and repair of the.
Binding of two zinc finger nuclease monomers to two. We have investigated the ability of singlestrand specific sss nucleases from different sources to cleave single base pair mismatches in heteroduplex dna. Zinc finger nuclease an overview sciencedirect topics. What is the best way to deliver the zfn and the repair template into the cells. The cleavingdomains are fok 1, which make a break in the dna in both. Zinc finger nucleases are designed proteins that fuse a zinc finger dna binding domain. The technique can identify one or more rna molecules of known sequence even at low total concentration.
A zfn is a hybrid molecule that couples the dna binding domain of a zincfinger protein with the dnacleaving nuclease domain of the restriction endonuclease foki. The goal of this study thus, was constructing a database of zinc finger. Only a few percent of the injected eggs giving rise totransgenic animals. We engineer zfns for precision, efficiency, and specificity, which we believe are the critical parameters for a therapeutic genome editing technology. The dsb is induced by a zinc finger nuclease zfn4,5, which is a designed, sequence specific endonuclease that can be customized to cleave a userchosen dna target. Enhancing zincfingernuclease activity with improved. Our knowledge of the oglycoproteome nacetylgalactosamine galnac type is highly limited. Label license agreement zinc finger nuclease zfn 2. In this approach, human c2bbe1 cells a subclone of caco. A zinc finger nuclease, transcription activatorlike effector nuclease, or laglidadg homing endonuclease transgene is transformed into. The mrna or dna encoding a zincfinger nuclease zfn, a transcription activatorlike effector nuclease talen, or a clustered regularly interspaced short palindromic repeatcrisprassociated crisprcas system, and a repair template could potentially be introduced into cells using nonviral vectors to carry out gene editing. Zincfinger nucleases zfns are synthetic enzymes that can be designed to generate targeted doublestranded breaks in dna, giving the scientific community a way to effectively stimulate genomic changes. This article describes the process for the design, manufacture, validation and implementation of the compozr custom zfn service.
Both authors contributed equally to the writing of this article. Until recently, the generation of animals with gene targeted manipulations has been accomplished by homologous recombination hr in embryonic stem es cells or cloning through. Plants developed by zfn3 european food safety authority. When 2f rna is incorporated into an antisense dna oligo resulting in a 2f rnadna chimeric, the tm of its duplex with rna increases relative to that formed by an unmodified antisense dna oligo by 1. Use of zinc finger nuclease technology to knock out efflux. Our zinc finger nuclease zfn technology is based on a naturally occurring class of proteins called zinc finger dnabinding proteins zfps, which recognize and bind to specific sequences of dna. Wolfe1,2, 1program in gene function and expression, university of massachusetts medical school, worcester, ma. Use of zinc finger nuclease technology to knock out efflux transporters in c2bbe1 cells. Binding of two zinc finger nuclease monomers to two specific sites is required for effective doublestrand dna cleavage. This new technology opens up revolutionary possibilities in agriculture, animal husbandry and molecular medicine.
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